The invention relates to methods for measuring the function of lymphocytes and their responses to mitogens or specific antigens. The methods are suitable for measurement of the responses of T lymphocytes when they are a subpopulation of cells, and also for measuring the function of specific subsets of T lymphocytes, each subpopulation or subset of a subpopulation having characteristic determinants on their cell surface. The invention also relates to test kits used in performing such methods. The methods of the invention facilitate screening of complex biological fluids, such as whole blood, by means of incubating a sample of the fluid with a mitogen or antigen, separating the selected subset of interest, e.g., via affinity separation, and detecting the presence of an internal cellular component, advantageously ATP, that is increased as a result of the response.
The immune system is central to control of infectious diseases and cancer. Lymphocytes, a class of white blood cells, are critical cell types that are responsible for the activities of the immune system. Lymphocytes are divided into two major categories, T lymphocytes and B lymphocytes. Overall assessment of the function of the immune systems and, in particular, lymphocytes is important in assessment of immunodeficiency caused by: genetic factors, infectious disease such as (HIV), drugs following transplantation, stress, aging, or nutritional deprivation.
Lymphocytes express receptors on the cell surface that bind with specific antigens or epitopes. Exposure to the antigen results in expansion in the population of the lymphocytes that are reactive to that antigen. Measurement of the response of the immune system to a specific antigen can be useful in diagnosis of infectious disease, hypersensitivity to certain agents, exposure to immunologically reactive drugs, or response to vaccination.
The function of B lymphocytes or their response to specific antigen can be assessed by measuring the level of specific antibody in bodily fluids such as blood, saliva or urine. The function of T lymphocytes or their response to specific antigens is more difficult to measure. Measurement of the functions of T lymphocytes or T cells is complicated by a number of factors. First, there are several different subsets of T cells with different functions. These subsets have been classified in part by the expression of characteristic cell surface markers and in part by a variety of functional assays including measurement of cytokines. Second, T cells respond to antigens only when they are presented by other cells in the context of major histocompatibility antigens on the surface of the presenting cell. Third, many of the functions of T cells depend on cell-cell contact with effector cells or the functions are fairly localized. Current methods for measuring immune function are tedious, time consuming, and poorly adapted to the clinical laboratory setting.
Methods that are currently used for measurement of Immune function include: methods based on counting the number of T cells or different subsets; methods based on measuring the proliferation of lymphocytes, methods based on measurement of cytotoxic activity or secretion of cytokines, and methods used in vivo such as skin tests and adoptive transfer. These methods are described in detail in the literature (see for example Groeneveld et al., Journal of the International Federation of Clinical Chemistry, 6: 84-94; 1994; Clough and Roth, JAVMA 206:1208-1216, 1995).
The methods most commonly used in the clinical laboratories are based on counting the number of T cells or subsets. A variety of techniques have been described including immunofluorescence microscopy, immunocytochemistry, enzyme immunoassay, and flow cytometry. Flow cytometry, in particular, is widely used in clinical laboratory settings. Flow cytometry is particularly useful in measurement of subsets of interest within a complex population of cells. For example, U.S. Pat. No. 4,727,020 to Recktenwald describes the use of two fluorescent channels to detect cells in a subpopulation specifically labeled with two different immunofluorescent agents. U.S. Pat. No. 4,284,412 to Hansen, et al. describes the use of fluorescence channels to detect forward and right angle light scatter of cells of different subpopulations in blood. Major disadvantages of flow cytometry are that it requires complex and expensive equipment, each sample must be run and analyzed individually and the results require interpretation and are frequently not repeatable. These disadvantages are particularly acute in a clinical laboratory which must process multiple patient specimens daily and where the need for consistent and reliable results is extremely important.
U.S Pat. No. 5,385,822 to Melnicoff et. al. and U.S. Pat. No. 5,374,531 to Jensen disclose alternative methods to flow cytometry for counting the number of lymphocytes or of a subset of lymphocytes within a mixed population of cells. The methods described in these patents involve coupling a detectable reporter substance to the bio-membrane or incorporating the reporter substance into the cell, then separating the subset or population of interest and detecting the reporter substance. These methods utilize affinity separation to isolate populations of interest from a complex mixture of cells. This technique offers improvements over flow cytometry but it is still based on cell counting techniques.
The major difficulty with all cell counting techniques is that they do not measure the function of specific cells or their responses to specific antigens or mitogens. Cells that respond to mitogens or antigens have unique cell surface markers found only on the responding cells. Methods for counting the number of cells exhibiting these markers have been described but these methods are relatively insensitive due to the fact that the responding cells are generally a small fraction of the total population. These methods are also tedious and subject to poor reproducibility.
Direct measurement of responses of lymphocytes have included lymphoproliferation assays, cytotoxicity assays, and measurement of cytokines. In general, these methods require separation of white cells from the original sample followed by incubation with antigen or mitogen. Measurement of the function of specific subsets of lymphocytes requires extensive manipulations prior to the assay. The requirement for antigen presenting cells then means that additional cells have to be added back to the culture. Lymphoproliferation assays are based on division of responding cells and are typically performed using radioactive isotopes. Because they evaluate the division of a small population of cells and require tissue culture, the assays take 3-10 days and are subject to significant variability based on the specific technique and the reagents used in the assay. Cytotoxic tests also require significant cell manipulation and are similarly highly variable depending on the specific conditions used. Cytokine assays can also be performed, but require many steps and separation of subsets of interest prior to stimulating the cells. U.S. Pat. No. 5,344,755 to McMicheals describes a modification of the cytotoxic assay based on initial immunomagnetic separation of T lymphocytes, but this method still requires extensive manipulation of effector cells. U.S. Pat. No. 5,344,755 provides an example of use of cytokine measurements to assess immune status in HIV positive patients but is tedious and requires multiple steps. These methods have required separation of critical cell types, long incubation times, and in some cases use of radioactive substances. For these reasons, these methods have not been suitable for clinical applications.
Affinity separation of cells using protein-coated magnetic particles or other types of solid supports such as polystyrene particles is known and is used as part of several of the methods cited above, see U.S. Pat. Nos. 5,374,531, 5,385,822, and 5,344,755. Various methods for sorting biological populations via affinity separations on solid supports have been described in the patent literature and elsewhere. See, for example, U.S. Pat. Nos. 3,970,518, 4,710,472, 4,677,067, 4,666,595, 4,230,685, 4,219,411, 4,157,323; see also, E. T. Menz, et al., Am. Biotech. Lab. (1986); J. S. Kemshead et al., Molec. Cell. Biochem., 67:11-18 (1985); T. Leivestag et al., Tissue Antigens, 28:46-52 (1986); and J. S. Berman et al., J. Immunol., 138: 2100-03 (1987). In performing such methods, a binding molecule (e.g., monoclonal antibody) is typically conjugated to the solid supports such as the magnetic particles or plastic beads, and added to a test sample under conditions causing binding to a characteristic determinant on the analyte of interest. The cells complexed with the solid support are then separated from the uncomplexed cells by exposure to a magnetic field or filtration or other method depending on the nature of the solid support. The use of this technology to separate certain subpopulations of lymphocytes from bone marrow cells prior to transplantation and to eliminate post-transplantation graft vs. host reaction, has also been reported. See A. Butturini et al., Prog. Bone Marrow Transpl. 4:13-22 (1987). Other reported uses of this technology include the separation of tumor cells (see: Kemshead et al., B.J. Cancer 54:771-78 (1986)) and the separation of lymphocyte subpopulations for subsequent functional evaluation.
The problems that arise when the lymphocytes are first separated by magnetic or other solid phase affinity techniques and then used for functional assays, are that the interaction of the lymphocyte with the binding molecule can itself induce functional changes in the lymphocyte that may obscure later changes that are to be measured. In addition, the accessory cells required for response of the T cells may no longer be present especially if a specific subset of cells are isolated. In addition, isolated cells are removed from the native environment and it is difficult to maintain the sterility of the sample required for further tissue culture.
The subject invention provides a convenient, reliable, and relatively rapid method for analyzing the function of various sets or subsets of lymphocytes. The method of the invention involves incubating a population of cells with a mitogen or antigen, separating the desired subset of cells by means of the interaction of a specific binding reagent that is attached to the solid phase with a cell surface determinant that is present on the cell subset of interest, lysing of the separated cells, and measuring an intracellular component that is increased if the cells have responded to the stimulus.
In an advantageous embodiment of the invention, the functional activity of a set or subset of lymphocytes which is distinguished by a characteristic cell surface determinant and which is contained within a mixed cell population, is measured by: exposing the sample to a mitogen or antigen, incubating the sample for a period of time, binding the set or subset of lymphocytes to a solid support through the interaction of the cell surface determinant and a specific binding substance which is linked to the solid support, washing the cells to remove any unbound cells as well as potentially interfering substances in the media, lysing the cells, and detecting the ATP in the solution. The results obtained can be compared against a known standard. Alternatively, the sample can be divided into two or more parts with at least one of the parts being incubated without addition of any stimulant, while the second part is incubated with addition of an antigen or mitogen.
One aspect of the invention is the determination of the response of lymphocytes to a mitogen. In this case, the response of a set of lymphocytes to a mitogen is a general measure of immune function. This application is of particular significance in the measurement of the effects of immunosuppressive drugs or agents. In this case, the responsiveness of an entire set of lymphocytes such as the T lymphocytes can be determined. Alternatively, the effect of a virus such as HIV can be assessed by evaluating the response to mitogens of a subset of T lymphocytes that express the CD4 cell surface determinant.
Another aspect of the invention is the determination of the response of lymphocytes to antigens that might include infectious agents, drugs, chemicals, autoantigens, or tumor antigens. This aspect of the invention is particularly important in monitoring the exposure of an individual to an infectious disease or an agent or to diagnosis of hypersensitivity, autoimmune disease or cancer. This aspect of the invention is also useful in monitoring vaccine efficacy and in assessment of immunotoxicity of chemicals, drugs, and industrial compounds.
Another aspect of the invention is that the response of T lymphocytes from various subsets including functional or differentiation based subsets to a mitogen or antigen can be assessed based on the expression of different determinants specific for a functional, differentiation, or activation marker on the cell surface. In this aspect of the invention, an antigen is added to a sample and incubated for a period of time. Following the incubation, the cell subset of interest is isolated by binding the cells to a solid support through a determinant on the cell surface. The cells are washed and lysed, and the level of an intracellular component that increases as a result of the activation is measured.
The method of the invention is highly sensitive due to the measurement of an intracellular component whose level increases rapidly following lymphocyte exposure to mitogen or antigen, for example ATP, other metabolic intermediates such as NADP, or proteins involved in cell cycle regulation such as PCNA. In an advantageous embodiment, the level of ATP is measured utilizing the bioluminescent reaction of luciferin:luciferase. Bioluminescent measurement of ATP is a highly sensitive measure.
A further aspect of the invention is that the total time required for the method is generally 6-72 hours and typically 18-24 hours. The relatively short time period is an advantage in comparison to the current methods which requires 3-10 days to complete.
In one embodiment of the invention, test kits are provided for performing the methods of the invention. Test kits typically contain the antigen or mitogen, the solid state with appropriate binding substance, the reagents required for detection of ATP levels, appropriate diluent and wash solutions, standards or instructions for preparing the same, and optionally, other accessories such as test tubes, magnetic separators, washers, and transfer pipettes, which are useful in carrying out the methods of the invention.